To generate C-terminal fusion proteins, we take advantage of the Gateway cloning system to simultaneously clone multiple fragments (Hope et al. 2004; Sasaki et al. 2005). In this case, we begin by generating the proper ENTRY clones (we hope that as more zebrafish labs use this approach, more ENTRY clones will be available, obviating the need to generate your own). This entails PCR amplifying the ORF of interest without a stop codon as well as a 3' tag (in this case EGFP) using the appropriate att-containing PCR primers. Subsequently, we perform BP reactions with these PCR products into the corresponding pDONR vectors to generate ENTRY clones. < back|next >